Csm710

The PLA was performed to confirm the interaction of Trio with Myh9 according to the manufacturer's protocol (Sigma Aldrich #DUO92102). In short, NCCs were seeded onto slides in a 24-well plate at a density of 2 × 10⁴ cells/well, washed 2 times with PBS and fixed in 4% PFA for 20-30 min and then permeabilized in 0.5% Triton X-100 for 20 min, washed 2 times with PBS, blocked with blocking reagents for 60 min at 37 ºC. After blocking, cells were incubated with primary antibodies against Trio (1:100, Abcam) and Myh9 (1:100, proteintech) or RAC1 (1:100, Cytoskeleton) and β-catenin (1:100, Cell Signaling Technology) overnight at 4 ºC, followed by corresponding secondary antibodies conjugated with PLA probes for 60 min at 37 ºC. Cells were then incubated with ligation reagent for 30 min and with amplification reagent for 100 min at 37 ºC in dark. Next, cells were washed three times with wash buffer and incubated with a mounting medium with DAPI for 15 min in dark. Finally, Duolink and DAPI signals were detected using confocal microscopy (ZEISS CSM710).

NCCs were seeded on glass coverslips to reach 90% confluence. Followed by starvation in serum-free medium for 24 h, NCCs were wounded with a 10 uL pipette tip and washed by PBS. Subsequently, cells were incubated in a medium with 5% FBS for 24 h, and then fixed in 4% PFA, and incubated with GM130 (#ab2899, Abcam) at 4 ºC overnight. The next day, the cells were incubated with secondary antibodies to recognize GM130 and phalloidin-FITC to recognize F-actin and then incubated with DAPI to detect Nuclei. The cells were then visualized with a confocal microscope (ZEISS CSM710). The position of the Golgi apparatus and nucleus reorientation was measured as previously reported 74 (link). Cdc42 specific activator Bradykinin was purchased by SIGMA, Munich, Germany.